Methods of detecting and/or measuring a nucleic acid wherein an enzyme produces a labeled nucleic acid fragment in a cleavage reaction are known in the art.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 disclose a method of cleaving a target DNA molecule by incubating a 5′ labeled target DNA with a DNA polymerase isolated from Thermus aquaticus (Taq polymerase) and a partially complementary oligonucleotide capable of hybridizing to sequences at the desired point of cleavage. The partially complementary oligonucleotide directs the Taq polymerase to the target DNA through formation of a substrate structure containing a duplex with a 3′ extension opposite the desired site of cleavage wherein the non-complementary region of the oligonucleotide provides a 3′ arm and the unannealed 5′ region of the substrate molecule provides a 5′ arm. The partially complementary oligonucleotide includes a 3′ nucleotide extension capable of forming a short hairpin either when unhybridized or when hybridized to a target sequence at the desired point of cleavage. The release of labeled fragment is detected following cleavage by Taq polymerase.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 disclose the generation of mutant, thermostable DNA polymerases that have very little or no detectable synthetic activity, and wild type thermostable nuclease activity. The mutant polymerases are said to be useful because they lack 5′ to 3′ synthetic activity; thus synthetic activity is an undesirable side reaction in combination with a DNA cleavage step in a detection assay.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 disclose that wild type Taq polymerase or mutant Taq polymerases that lack synthetic activity can release a labeled fragment by cleaving a 5′ end labeled hairpin structure formed by heat denaturation followed by cooling, in the presence of a primer that binds to the 3′ arm of the hairpin structure. Further, U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 teach that the mutant Taq polymerases lacking synthetic activity can also cleave this hairpin structure in the absence of a primer that binds to the 3′ arm of the hairpin structure.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 also disclose that cleavage of this hairpin structure in the presence of a primer that binds to the 3′ arm of the hairpin structure by mutant Taq polymerases lacking synthetic activity yields a single species of labeled cleaved product, while wild type Taq polymerase produces multiple cleavage products and converts the hairpin structure to a double stranded form in the presence of dNTPs, due to the high level of synthetic activity of the wild type Taq enzyme.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 also disclose that mutant Taq polymerases exhibiting reduced synthetic activity, but not wild type Taq polymerase, can release a single labeled fragment by cleaving a linear nucleic acid substrate comprising a 5′ end labeled target nucleic acid and a complementary oligonucleotide wherein the complementary oligonucleotide hybridizes to a portion of the target nucleic acid such that 5′ and 3′ regions of the target nucleic acid are not annealed to the oligonucleotide and remain single stranded.
There is a need in the art for a method of generating a signal that does not require formation of a cleavage structure and does not require an additional cleavage step.
Methods of detecting and/or measuring a nucleic acid in the absence of a cleavage step are known in the art.
U.S. Pat. Nos. 6,103,476 and 5,925,517 teach unimolecular and bimolecular hybridization probes for the detection of nucleic acid target sequences, comprising a target complement sequence, and an affinity pair holding the probe in a closed conformation in the absence of target sequence. According to the method of the invention of these patents, a probe comprises either a label pair that interacts when the probe is in the closed conformation or, for certain unimolecular probes, a non-interactive label.
U.S. Pat. Nos. 6,103,476 and 5,925,517 teach generation of a signal as a result of hybridization of the probe and target sequences and a conformational change in the prot such that the probe shifts to an open conformation. The conformational change in the probe that occurs as a result of hybridization, is detectable due to reduced interaction of the label pair or by detecting a signal from a non-interactive label.
U.S. Pat. No. 6,150,097 relates to nucleic acid hybridization probes (bimolecular, according to one embodiment) that are labeled with a non-FRET pair of chromophores and undergo a conformational change as a result of interacting with a target. This patent teaches generation of a signal as a result of hybridization of the probe and target and a conformational change in the probe that alters the distance between the label pair. The conformational change in the probe that occurs as a result of hybridization, is detectable due to increased or reduced interaction of the label pair.
None of these patents teach generation of a signal as a result of hybridization of the probe to the target and subsequent dissociation of at least one subunit of the probe. Furthermore, none of these patents teach generation of a signal as a result of hybridization of the probe to the target, displacement of at least one subunit of the probe by the synthetic activity of a polymerase and subsequent dissociation of at least one subunit of the probe. None of these patents teach a probe comprising a binding moiety that is inaccessible unless the probe has hybridized to a target and at least the subunit of the probe comprising the binding moiety dissociates from at least a second subunit of the probe. Additionally, none of these patents teach a probe comprising a binding moiety that is inaccessible unless the probe has hybridized to a target and undergone a conformational change wherein at least the subunit of the probe comprising the binding moiety dissociates from at least a second subunit of the probe.
There is a need in the art for a method of generating a signal wherein the generated signal is released from a target nucleic acid and can be measured in the absence of the target nucleic acid. A method of generating a signal wherein the signal is released from the target nucleic acid offers the advantage of an increased signal. For example, by completely separating two members of an interactive label pair (i.e., by dissociating two subunits of a probe, each of which individually comprises one member of the interactive label pair), the intensity of the signal is increased and the background is decreased.
There is also a need in the art for a simplified method of generating a signal that does not require multiple steps, including a cleavage step.
There is a need in the art for a method of generating a signal that utilizes a probe having at least two subunits wherein the first subunit comprises a binding moiety and further comprising secondary structure wherein some or all of the self-complementary regions of the probe that anneal to form the secondary structure are melted when the probe hybridizes with a target nucleic acid, thereby reducing non-specific binding of the probe to the target, and increasing the specificity of the assay.
U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780 also disclose a method of cleaving a labeled nucleic acid substrate at naturally occurring areas of secondary structure. According to this method, biotin labeled DNA substrates are prepared by PCR, mixed with wild type Taq polymerase or CleavaseBN (a mutant Taq polymerase with reduced synthetic activity and wild type 5′ to 3′ nuclease activity), incubated at 95° C. for 5 seconds to denature the substrate and then quickly cooled to 65° C. to allow the DNA to assume its unique secondary structure by allowing the formation of intra-strand hydrogen bonds between the complementary bases. The reaction mixture is incubated at 65° C. to allow cleavage to occur and biotinylated cleavage products are detected.
The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat. Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific DNA sequences, using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment whose termini are defined by the 5′ ends of the primers. PCR is reported to be capable of producing a selective enrichment of a specific DNA sequence by a factor of 109. The PCR method is also described in Saiki et al., 1985, Science, 230:1350.
While the PCR technique is an extremely powerful method for amplifying nucleic acid sequences, the detection of the amplified material requires additional manipulation and subsequent handling of the PCR products to determine whether the target DNA is present. It is desirable to decrease the number of subsequent handling steps currently required for the detection of amplified material. An assay system, wherein a signal is generated while the target sequence is amplified, requires fewer handling steps for the detection of amplified material, as compared to a PCR method that does not generate a signal during the amplification step.
U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose a PCR based assay for releasing labeled probe comprising generating a signal during the amplification step of a PCR reaction in the presence of a nucleic acid to be amplified, Taq polymerase that has 5′ to 3′ exonuclease activity and a 5′, 3′ or 5′ and 3′ end-labeled probe comprising a region complementary to the amplified region and an additional non-complementary 5′ tail region. U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose further that this PCR based assay can liberate the 5′ labeled end of a hybridized probe when the Taq polymerase is positioned near the labeled probe by an upstream probe in a polymerization independent manner, e.g. in the absence of dNTPs.
There is a need in the art for a method of detecting or measuring a target nucleic acid that does not require multiple steps.
There is also a need in the art for a PCR process for detecting or measuring a target nucleic acid that does not require multiple steps subsequent to the amplification process.
There is also a need in the art for a PCR process for detecting or measuring a target nucleic acid that allows for concurrent amplification and detection of a target nucleic acid in a sample.
There is also a need in the art for a PCR process for detecting or measuring a target nucleic acid sample that does not require a cleavage step.